Trastuzumab has proven to be a very effective therapy for HER2-positive breast cancer, but de novo or acquired resistance limits its long-term value. New observations from our laboratory suggest several hypotheses on the mechanisms of resistance to trastuzumab and other therapies targeting the HER network. First, our results suggest that this resistance might stem from incomplete blockade of the signals generated from the various HER-family dimer pairs in the network input layer. Using a limited number of HER2- overexpressing xenograft models we have found that combined drug therapies designed to more completely block these heterodimers can overcome resistance to single agents and are even capable of eradicating many of these tumors in mice. Our preclinical models and preliminary patient data also suggest alternative mechanisms for resistance to HER-directed therapy that involve the estrogen receptor in some tumors and the MUC4 mucins in others. Here we propose a series of preclinical studies and an early phase clinical trial to begin to test these hypotheses. Specifically we will: 1) Confirm our preliminary data that resistance to single-agent HER targeted therapy can be overcome by various combinations of trastuzumab, lapatinib, and pertuzumab, designed to more completely block signaling from the HER network input layer, in a large panel of HER2-amplified breast cancer cell lines, and to identify and establish models resistant to these single and combined antiHER2 drugs for later studies; 2) Determine using these various preclinical HER2-positive models whether upregulation of ER or ER signaling to an alternative survival pathway can be induced by HER blockade as a resistance mechanism, and whether simultaneous targeting of ER and HER2 is then necessary for optimal treatment; 3) Investigate whether upregulation of MUC4 causes resistance to HER targeted therapy in our preclinical in vivo model system, thereby providing a new potential diagnostic and treatment target to investigate in human samples; 4) Lead a multi-institutional phase 2 neoadjuvant clinical trial of lapatinib combined with trastuzumab, with serial tissue sampling to assess molecular mechanisms of action and resistance, in order to begin to translate our exciting preclinical findings to patients. This work will facilitate new strategies to circumvent resistance to HER-targeted therapy for improved patient survival.